1. Field of the Invention
An aspect of the present invention relates to a method for producing a transformed earthworm using earthworm gonad-regeneration capability, a transformed earthworm produced by the method, and a method for producing a recombinant protein from a body fluid of the transformed earthworm.
2. Description of the Related Art
The conventional transformation method (transgenesis) is generally classified into somatic cell transformation and reproductive cell transformation. The somatic cell transformation is a method in which a new hereditary character appears in a disposition animal but is not delivered to the progeny. The reproductive cell transformation is a method in which a direct or the transformed cell induces a new gene to be transferred to a reproductive cell so that a new hereditary character is continuously expressed.
The reproductive cell transformation includes a pronuclear injection method, a method using a virus vector, a method using an embryonic stem cell, a nuclear transfer method, an in vitro fertilization method, and the like.
The pronuclear injection method is a method in which a transformant is built by placing a new gene in the nucleus of a fertilized egg and implanting on the uterine wall. In this method, the gene is randomly inserted into a chromosome in a cell division time of the implanted fertilized egg so that the character appears. Since the method is easiest and has high efficiency, the method is currently most frequently used in industry (Gordon J W, Ruddle F H, 1981. Science 214: 1244246; Hammer R E, Pursel V G, Rexroad C E, Jr., Wall R J, Bolt D J, Ebert K M, et al. 1985, Nature 315: 68083). While the new character is obtained with the expression of the new gene, one disadvantage is that it is difficult to control the expression degree due to random insertion and the correct result cannot be obtained.
The method using the virus vector is very similar to the pronuclear injection method, but includes a transformant that is built through a powerful infection method of viruses not by directly injecting a new gene but by producing a recombinant gene using a non-toxic processing virus vector. Since the method has very high gene injection efficiency, the method is used a gene therapy in humans. However, like the pronuclear injection method, it is difficult to control the expression degree due to random insertion (Jaenisch R, Fan H, Croker B, 1975. Proc Natl Acad Sci USA 72: 4008012; Robertson E, Bradley A, Kuehn M, Evans M, 1986. Nature 323: 44548).
The method using the embryonic stem cell is a method which, if an embryonic stem cell transformed by introducing a new gene to a blast cell of the embryonic stem cell having totipotency is transplanted to a new fertilized egg, the embryonic stem cell transplanted in a fetus development process is specialized as a reproductive cell so that a transformed animal is born. Since a specific hereditary part of the transformed animal produced by the method is controlled, the method is widely used in industrial and medical fields. On the other hand, the method takes more time, costs more than the other methods, and is not currently built for other animals than rodents (Martin G R, 1981. Proc Natl Acad Sci USA 78: 7634638; Thomas K R, Capecchi M R, 1987. Cell 51: 50312; Nagano M, Brinster C J, Orwig K E, Ryu B Y, Avarbock M R, Brinster R L, 2001. Proc Natl Acad Sci USA 98: 130903095).
The nuclear transfer method is a method well known through the cloned lamb Dolly, in which the nucleus of an unfertilized egg is removed, and the nucleus of a donor cell is transplanted to the unfertilized egg having the nucleus removed therefrom, so that the egg with the transplanted nucleus is developed in the interior of the body of a surrogate (animal) to an entity. In this method, from fertilized egg originated early cells to somatic cells can be used almost without limit as a nuclear donor cell, and a large number of transformed animals can be cloned and produced at a time. On the other hand, the method has problems to be solved, including very low production efficiency, delivery of stillborn and deformed children, and the like (Willadsen S M, 1986. Nature 320: 635; Schnieke A E, Kind A J, Ritchie W A, Mycock K, Scott A R, Ritchie M, Wilmut I, et al., 1997. Science 278: 2130133; Cibelli J B, Stice S L, Golueke P J, Kane J J, Jerry J, Blackwell C, et al., 1998. Science 280: 1256258).
The in vitro fertilization method is a familiar to us as a test-tube baby, in which the fertilization of a sperm and an ovum is performed in vitro and the fertilized egg is then transplanted to a mother to be fertilized. Transformed animals are produced using the method. If the ovum is fertilized with the sperm having a new gene attached to a head part thereof before the external fertilization is performed, a new character is expressed by the new gene, so that it is possible to produce a transformed animal. The method is most simple among the conventional transformed animal producing methods, and does not require expensive equipment. On the other hand, there are a few successful cases, and only random gene transformation is possible (Brackett B G, Baranska W, Sawicki W, Koprowski H, 1971. Proc Natl Acad Sci USA 68: 35357; Maione B, Lavitrano M, Spadafora C, Kiessling A A, 1998. Mol Reprod Dev 50: 40609; Rieth A, Pothier F, Sirad M A, 2000. Molecular Reproduction and Development 57: 33845; Celebi C, Guillaudeux T, Auvray P, Vallet-Erdtmann V, Jegou B 2003. Biol Reprod 68: 1477483).
However, all the methods described above require a large amount of time and cost in building transformants, and require expensive equipment or expertise.
Meanwhile, the earthworm is an oligochaete annelid called an earthworm, Pheretima communissima or the like. The earthworm is a hermaphrodite having male and female organs in one body, and lives on organisms in the soil of fertile damp areas everywhere. It is reported that over 2700 kinds of earthworms are distributed all over the world, and over 60 kinds of earthworms lives in Korea. According to a medical book such as Dongeuibogam (Korea) or Bonchogangmok (China), since the earthworm contains a medical component, it is known that the earthworm has effects of hemolysis, removal of fever, etc., and is effective in killing parasites in intestines, fever removal, insanity, jaundice, seasonal epidemic, sore throat, etc. Also, it is known that some kinds of earthworms have regeneration capability. The regeneration of the earthworm is limited to a part posterior to an annulus, in which major organs are not contained, and the earthworms have a variety of regeneration capabilities according to their kinds. Some researchers have asserted a theory that organs prior to the annulus can be regenerated, but the biological mechanism and method of the theory have not yet been discovered. Studies on a transgenesis technique using the regeneration capability of earthworms have not been conducted so far.
Therefore, developing a transgenesis technique using earthworm's regeneration capability is required.
While studying a method capable of improving the conventional transgenesis technique and simply building a transformant with little time and cost, the present inventors produced a transformed earthworm by cutting a front end part including a gonad of the earthworm, forming a regenerative blast cell through induction of regeneration and then injecting a recombinant gene expression vector into a part of the regenerative blast cell of the earthworm in the initial stage of regeneration. The inventors have completed the present invention by identifying that recombinant proteins can be produced from the body fluid of the transformed earthworm.